In our search for understanding the evolution of blood sucking by arthropods, we realize the methodology used has drastically changed in the past few years. Traditionally, the research process first identified a biological activity in saliva or salivary gland homogenates of a particular organism, and then proceeded to isolate that activity as a relatively pure entity to allow its molecular identification. In the case the activity derived from a protein, peptide fingerprinting allowed the design and use of nucleotide probes to clone the coding mRNA (in the form of a cDNA) and final identification of the peptide sequence;the clone also allowed the manufacture of recombinant protein for further studies. Nowadays, the process has reverted. cDNA libraries are constructed from salivary glands of blood sucking arthropods and mass sequenced. Bioinformatic analysis reveals the salivary transcriptome of these organisms, which contains many unique protein families with unknown properties. We then proceed to select clones for expression, bioassay screening and characterization. Accordingly, there are two processes used in our lab, first, the construction and analysis of salivary gland cDNA libraries, and second, the recombinant expression and characterization of these proteins. We have also been developing bioinformatic capabilities in the form of specific software to help direct our studies. Sialotranscriptome discovery projects: Because host hemostasis (the physiological process that prevents blood loss, consisting of platelet aggregation, blood clotting and vasoconstriction) is a complex and redundant phenomenon, the salivary glands of blood sucking arthropods consist of a magic potion with diverse chemicals that in a redundant way counteract host mechanisms to prevent blood loss, allowing the fast acquisition of a meal. Salivary transcriptome made in the past few years indicate that the magic potion consists of 70-100 different proteins in the case of mosquitoes, for example, to over 400 in the case of ticks (Ticks feed for several days and have to disarm host immune reactions, in addition to the hemostatic system). Transcriptome studies also show that the salivary proteins of blood sucking arthropods are at a very fast pace of evolution, perhaps explaining why every genera studied so far has several unique protein families. Indeed there are unique proteins found at the subgenus level. Given we can now describe in detail the sialotranscriptome (from the Greek word sialo = saliva) of a single organism, we can ask now what is the universe of salivary proteins associated to blood feeding, the so called sialoverse. There are near 19,000 species of blood sucking arthropods in 500 different genera. If we find (minimally) 5 novel protein families per genus (within the 70-500 proteins in each sialome), there are at least 2,500 novel proteins to be discovered, each one with an interesting pharmacological property. We have so far explored less than a dozen genera of blood sucking arthropods, and it is our goal to extend sialotranscriptome discovery to map this pharmacological mine for future studies, and in the process learn the paths taken by genomes in their evolution to blood feeding, and identify proteins with pharmacological and vaccine potential. In the current fiscal year (2009), we produced a total of 14 papers and one patent application. Five of the papers describe sialomes, including the sialome of the black fly Simulium vittatum (1), the first so far produced for this family of flies, of the South American malaria vector Anopheles darlingi (2), of larval An. gambiae mosquitoes (3), and of the stable fly, Stomoxys calcitrans (4), the first from the fly suborder Brachycera, and of a soft tick Ornithodoros coriaceus (5). Several new protein families were discovered in the sialomes of the black fly and the stable fly that awaits functional characterization. The An. darlingi sialome allowed understanding of the fast evolution of mosquito salivary proteins, including the differentiation of families between the sub families anopheline and culicine. The sialome of larval mosquitoes, which do not blood feed, allow for comparison with those of the adult female and identification of unique proteins that are associated with blood feeding. Finally, the sialome of O. coriaceus allowed for the identification of families of proteins common to soft ticks and determination of the fast divergence of these protein families. Functional sialomic studies: We advanced our knowledge regarding the function of several salivary proteins, as well as discovering novel salivary properties. One of the most abundant proteins of the mosquito vector of Yellow Fever was crystallized and its function revealed as a protein scavenger of serotonin and inflammatory leukotrienes (6). A salivary cysteinyl protease inhibitor of the Lyme disease vector, Ixodes scapularis, was shown to inhibit dendritic cells and to have immunosuppressive activity (7). The gSG6 protein of An. gambiae has been immunologically characterized as a good marker of vector exposure (8). A review article on the analysis of tick salivary proteins was also written (9). Expertise capabilities spin off: Our bioinformatic capability lead to collaboration with diverse studies, leading to the publishing of software to analyze transcriptomes (10), which is being widely used;for helping annotating secreted proteins of parasitic worms (11);to identify immune-related genes in the malaria vector Anopheles gambiae (12), and to model the evolution of transposable elements in mosquitoes (13). Our collaboration with Dr. Warwick Britton, lead to the identification in the genome of Mycobacterium tuberculosis of a family of cutinase proteins that has vaccine potential, reported in 2008. Now we report biochemical properties of these recombinant enzymes and enzyme active site identification (14). A patent application has been submitted for the use of a tick anticlotting agent as a suppressor of metastasis for cancer treatment (15).